Previous studies on testicular Leydig cell function have been performed in vivo or in dispersed cell suspension and have concentrated primarily on the role of gonadotropin in Leydig cell function. In the present study we propose to use a serum-free hormone supplemented culture system to study the effects of a variety of hormones, transport proteins, and growth factors on several parameters of Leydig cell function under defined conditions. A non-tumorigenic mouse clonal cell line, TM3, will be used as the most precisely defined cell culture system. In addition, primary cultures of Leydig cells will be studied from the rat, mouse and pig in order to have a cross-species comparison of the response of an established cell line and primary cultures from the same species. Parameters to be measured include cell survival and growth, steroid secretion (basal and hCG-stimulated), prostaglandin secretion, hormone receptor levels (androgen, progestin, insulin, EGF, hCG) and secreted protein. A set of hormone-supplements will be defined which are optimal for each function. These are expected to be overlapping but not identical. These studies should advance our understanding of the complex interrelationships of the hormonal control of Leydig cell function.